Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia

Dissecting the Extracellular Complexity of Neuromuscular Junction Organizers

Synapse formation is a very elaborate process dependent upon accurate coordination of pre and post-synaptic specialization, requiring multiple steps and a variety of receptors and signaling molecules. Due to its relative structural simplicity and the ease in manipulation and observation, the neuromuscular synapse or neuromuscular junction (NMJ)—the connection between motor neurons and skeletal muscle—represents the archetype junction system for studying synapse formation and conservation. This junction is essential for survival, as it controls our ability to move and breath. NMJ formation requires coordinated interactions between motor neurons and muscle fibers, which ultimately result in the formation of a highly specialized post-synaptic architecture and a highly differentiated nerve terminal. Furthermore, to ensure a fast and reliable synaptic transmission following neurotransmitter release, ligand-gated channels (acetylcholine receptors, AChRs) are clustered on the post-synaptic muscle cell at high concentrations in sites opposite the presynaptic active zone, supporting a direct role for nerves in the organization of the post-synaptic membrane architecture. This organized clustering process, essential for NMJ formation and for life, relies on key signaling molecules and receptors and is regulated by soluble extracellular molecules localized within the synaptic cleft. Notably, several mutations as well as auto-antibodies against components of these signaling complexes have been related to neuromuscular disorders. The recent years have witnessed strong progress in the understanding of molecular identities, architectures, and functions of NMJ macromolecules. Among these, prominent roles have been proposed for neural variants of the proteoglycan agrin, its receptor at NMJs composed of the lipoprotein receptor-related protein 4 (LRP4) and the muscle-specific kinase (MuSK), as well as the regulatory soluble synapse-specific protease Neurotrypsin. In this review we summarize the current state of the art regarding molecular structures and (agrin-dependent) canonical, as well as (agrin-independent) non-canonical, MuSK signaling mechanisms that underscore the formation of neuromuscular junctions, with the aim of providing a broad perspective to further stimulate molecular, cellular and tissue biology investigations on this fundamental intercellular contact

Export of functional Streptomyces coelicolor alditol oxidase to the periplasm or cell surface of Escherichia coli and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.

Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors

Here we report corin, a synthetic hybrid agent derived from the class I HDAC inhibitor (entinostat) and an LSD1 inhibitor (tranylcypromine analog). Enzymologic analysis reveals that corin potently targets the CoREST complex and shows more sustained inhibition of CoREST complex HDAC activity compared with entinostat. Cell-based experiments demonstrate that corin exhibits a superior anti-proliferative profile against several melanoma lines and cutaneous squamous cell carcinoma lines compared to its parent monofunctional inhibitors but is less toxic to melanocytes and keratinocytes. CoREST knockdown, gene expression, and ChIP studies suggest that corin's favorable pharmacologic effects may rely on an intact CoREST complex. Corin was also effective in slowing tumor growth in a melanoma mouse xenograft model. These studies highlight the promise of a new class of two-pronged hybrid agents that may show preferential targeting of particular epigenetic regulatory complexes and offer unique therapeutic opportunities

Menstrual mood disorders are associated with blunted sympathetic reactivity to stress

AbstractObjectiveFew studies have directly compared women with a menstrually related mood disorder (MRMD) with women who have suffered from depression for stress reactivity phenotypes. It is unclear whether blunted responses to stress in women with a MRMD reflect a unique phenotype of MRMDs or may be explained by a history of depression.MethodsWe assessed cardiovascular reactivity to stress in four groups: 1) Women with a MRMD without a history of depression (n=37); 2) women with a MRMD plus a history of depression (n=26); 3) women without a MRMD and without a history of depression (n=43); and 4) women without a MRMD but with a history of depression (n=20).ResultsWomen with a MRMD showed blunted myocardial (heart rate and cardiac index) reactivity to mental stress compared to non-MRMD women, irrespective of histories of depression. Hypo-reactivity to stress predicted greater premenstrual symptom severity in the entire sample. Women with a MRMD showed blunted norepinephrine and diastolic blood pressure stress reactivity relative to women with no MRMD, but only when no history of depression was present. Both MRMD women and women with depression histories reported greater negative subjective responses to stress relative to their non-MRMD and never depressed counterparts.ConclusionOur findings support the assertion that a blunted stress reactivity profile represents a unique phenotype of MRMDs and also underscore the importance of psychiatric histories to stress reactivity. Furthermore, our results emphasize the clinical relevance of myocardial hypo-reactivity to stress, since it predicts heightened premenstrual symptom severity

Non-coding RNA Expression, Function, and Variation during Drosophila Embryogenesis

Long non-coding RNAs (lncRNAs) can often function in the regulation of gene expression during development; however, their generality as essential regulators in developmental processes and organismal phenotypes remains unclear. Here, we performed a tailored investigation of lncRNA expression and function during Drosophila embryogenesis, interrogating multiple stages, tissue specificity, nuclear localization, and genetic backgrounds. Our results almost double the number of annotated lncRNAs expressed at these embryonic stages. lncRNA levels are generally positively correlated with those of their neighboring genes, with little evidence of transcriptional interference. Using fluorescent in situ hybridization, we report the spatiotemporal expression of 15 new lncRNAs, revealing very dynamic tissue-specific patterns. Despite this, deletion of selected lncRNA genes had no obvious developmental defects or effects on viability under standard and stressed conditions. However, two lncRNA deletions resulted in modest expression changes of a small number of genes, suggesting that they fine-tune expression of non-essential genes. Several lncRNAs have strain-specific expression, indicating that they are not fixed within the population. This intra-species variation across genetic backgrounds may thereby be a useful tool to distinguish rapidly evolving lncRNAs with as yet non-essential roles.Fil: Schor, Ignacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. European Molecular Biology Laboratory; AlemaniaFil: Bussotti, Giovanni. European Bioinformatics Institute; Reino UnidoFil: Maleš, Matilda. European Molecular Biology Laboratory; AlemaniaFil: Forneris, Mattia. European Molecular Biology Laboratory; AlemaniaFil: Viales, Rebecca R.. European Molecular Biology Laboratory; AlemaniaFil: Enright, Anton J.. European Bioinformatics Institute; Reino UnidoFil: Furlong, Eileen E. M.. European Molecular Biology Laboratory; Alemani

Enhancing clinical reasoning: Teaching thinking through debriefing

Session presented on: Monday, July 22, 2013: Purpose: How nurses are educated to think in practice is receiving well-deserved attention calling for the need for innovative and transformative strategies that guide nurses in the use of nursing knowledge and science. Guiding thinking to assist novice nurses to better infer meaning and apply clinical reasoning across contexts is an important teaching strategy. The overall purpose of this multi-site pilot study was to implement a debriefing strategy to determine its impact on clinical reasoning skills with undergraduate nursing students across four different colleges of nursing. This study replicates Dreifuerst\u27s original study using a reflective debriefing model to enhance clinical reasoning. Methods: A quasi-experimental, pre-test-post-test, repeated measure, research design was used in this pilot study to evaluate student nurses\u27 clinical reasoning in simulation using the Debriefing for Meaningful Learning (DML) model. A convenience sample of 30 second year baccalaureate nursing students was the purposive, target population for this research. Students participated in a geriatric nursing simulation using the National League for Nursing\u27s (NLN) Advancing Care Excellence for Seniors (ACES) simulation scenario. Clinical reasoning was measured through the Health Sciences Reasoning Test (HSRT). Results: The original study findings illustrated statistical significance in the change in HSRT scores between pre-test and post-test. Results of the current study are being compiled and analyzed. It is anticipated that the findings will demonstrate a positive change in clinical reasoning skills with use of the DML debriefing model. Conclusion: Enhancing the effectiveness of teaching strategies that guide students thinking and clinical reasoning becomes clear guide student thinking within the context of care. Teaching thinking through reflection and debriefing has been shown to create a positive change in clinical reasoning skills. Replication of these findings will support the utility of the reflective debriefing model adding to the nursing literature on effective teaching strategies that enhance clinical reasoning

Histories of major depression and premenstrual dysphoric disorder: Evidence for phenotypic differences

This study examined unique versus shared stress and pain-related phenotypes associated with premenstrual dysphoric disorder (PMDD) and prior major depressive disorder (MDD). Sympathetic nervous system (SNS) and hypothalamic-pituitary-adrenal (HPA)-axis measures were assessed at rest and during mental stress, as well as sensitivity to cold pressor and tourniquet ischemic pain tasks in four groups of women: (1) non-PMDD with no prior MDD (N=18); (2) non-PMDD with prior MDD (N=9); (3) PMDD with no prior MDD (N=17); (4) PMDD with prior MDD (N=10). PMDD women showed blunted SNS responses to stress compared to non-PMDD women, irrespective of prior MDD; while women with prior MDD showed exaggerated diastolic blood pressure responses to stress versus never depressed women, irrespective of PMDD. However, only in women with histories of MDD did PMDD women have lower cortisol concentrations than non-PMDD women, and only in non-PMDD women was MDD associated with reduced cold pressor pain sensitivity. These results suggest both unique phenotypic differences between women with PMDD and those with a history of MDD, but also indicate that histories of MDD may have special relevance for PMDD